Role of a pro-apoptotic Bcl-2 protein in the survival and death of leukemia cells

描述(申请人提供)：拟议的研究重点是人类NoxA的一种新的调节机制和生存功能，它是一种促凋亡的‘BH3-Only’Bcl-2家族蛋白，迅速成为血液系统恶性肿瘤的重要治疗靶点。激活的Noxa与其结合伙伴、支持生存的Bcl-2家族成员Mcl-1L相互作用，促进细胞色素c释放到胞浆中。NOXA/Mc1-1L相互作用在免疫系统细胞的存活和死亡中起着至关重要的作用，NOXA是葡萄糖剥夺诱导增殖中的人淋巴细胞和造血癌细胞系凋亡所必需的。这种仅有BH3的蛋白在大多数白血病中稳定和结构性地表达。初步观察确定，在营养物质和葡萄糖存在下，Noxa被激酶CDK5在丝氨酸残基上磷酸化，并鉴定出两个细胞质多蛋白质颗粒，隔离了修饰后的蛋白质。研究还表明，改良的、隔离的诺沙星可以调节葡萄糖代谢，并具有促进生存的功能。人类NOXA是翻译后调控的，它在白血病细胞中既起到促进生存的作用，又起到促凋亡的作用，这一假设将通过三个特定的目标进行检验。第一个目标是确定翻译后修饰的人NOXA如何在有氧糖酵解和细胞生存中发挥作用。CDK5和葡萄糖敏感的PI3K/Akt信号通路在NoxA这一新的代谢功能中的作用也将被研究。含有NOXA的胞质多蛋白颗粒将被提纯和鉴定；它们的功能相关性和在调节NOXA和有氧糖酵解中的作用将被确定。第二个目的是研究导致白血病细胞中NOXA促凋亡功能激活的凋亡刺激和信号通路/S。肿瘤抑制因子磷酸酶PHLPP2在激活白血病细胞中的NOXA和凋亡方面的作用将被确定。将特定的CDK5抑制剂与糖酵解抑制剂相结合的新策略将在白血病细胞系和患者样本上进行测试，稍后将在小鼠异种移植试验中得到验证。人类NOXA的非磷酸化突变体和多肽促进细胞凋亡的能力也将被确定。第三个目标是建立条件转基因小鼠模型，以研究人NOXA在白血病发生和葡萄糖代谢中的作用。利用Vav启动子-四环素反式激活因子(Vav-TTA)驱动的多西环素阻遏系统，人类BH3-Only蛋白将在造血系细胞中表达。该转基因模型将用于确定人类NOXA的表达是否促进白血病的发生，该蛋白的持续表达是否需要维持白血病的表型，以及NOXA在淋巴细胞中的结构性表达是否改变其代谢葡萄糖以产生能量的能力。初步研究表明，NOXA是一种典型的肿瘤抑制蛋白BH3-Only，在血液系统癌症中也具有生存功能。这项拟议的研究将确定在这些双重和相反的角色中参与调控NOXA的新的信号通路和中间体，并将导致针对淋巴和髓系白血病中这种蛋白的新治疗策略的开发。

DESCRIPTION (provided by applicant): The proposed research focuses on a novel regulatory mechanism and survival function for human Noxa, a pro- apoptotic 'BH3-only' Bcl-2 family protein, fast emerging as an important therapeutic target in hematological malignancies. Activated Noxa interacts with its binding partner, pro-survival Bcl-2 family member Mcl-1L, to facilitate the release of cytochrome c into the cytosol. Noxa/Mcl-1L interactions play a crucial role in the survival and death of cells of the immune system and Noxa is required for glucose deprivation-induced apoptosis of proliferating human lymphocytes and hematopoietic cancer cell lines. This BH3-only protein is stably and constitutively expressed in a majority of leukemias. Preliminary observations have determined that Noxa is phosphorylated on a serine residue by the kinase, Cdk5, in the presence of nutrients and glucose and have identified two cytosolic multi-protein particles that sequester the modified protein. Studies also suggest that modified, sequestered Noxa regulates glucose metabolism and has a pro-survival function. The hypothesis that human Noxa is post-translationally regulated and that it plays both pro-survival and pro-apoptotic roles in leukemia cells will be tested through three specific aims. The first aim is directed at determining how post-translationally modified human Noxa plays a role in aerobic glycolysis and cell survival. The contribution of Cdk5 and the glucose-sensitive PI3K/Akt signaling pathway to this novel metabolic function of Noxa will also be investigated. The cytosolic multi-protein particles containing Noxa will be purified and characterized; their functional relevance and role in regulating Noxa and aerobic glycolysis will be determined. The second aim is directed at investigating apoptotic stimuli and signaling pathway/s that lead to the activation of Noxa pro-apoptotic function in leukemia cells. The role of the tumor suppressor phosphatase, PHLPP2, in activating both Noxa and apoptosis in leukemia cells will be determined. Novel strategies that combine specific inhibitors of Cdk5 with inhibitors of glycolysis will be tested on leukemia cell lines and patient samples and later validated in murine xenograft assays. The ability of non- phosphorylatable mutants and peptides of human Noxa to promote apoptosis will also be determined. The third aim is directed at developing a conditional transgenic mouse model to investigate the role of human Noxa in leukemogenesis and glucose metabolism. The human BH3-only protein will be expressed in hematopoietic lineage cells, employing the Vav promoter-tetracyline transactivator (Vav-tTA)-driven doxycycline repressible system. The transgenic model will be utilized for determining whether expression of human Noxa promotes leukemogenesis, whether sustained expression of the protein is required for maintaining the leukemic phenotype, and whether constitutive expression of Noxa in lymphocytes alters their ability to metabolize glucose for energy generation. Preliminary studies show that Noxa, a canonical tumor suppressor BH3-only protein, also has a survival function in hematopoietic cancers. The proposed research will identify novel signaling pathways and intermediates involved in regulating Noxa in these dual and opposing roles, and should lead to the development of new therapeutic strategies for targeting this protein in lymphoid and myeloid leukemias.